The purpose of this application is to investigate and define how a coagulation cofactor modulates the substrate recognition, i.e. the specificity of its dependent enzyme. The enzymes which will be studied are factors VIIa, IXa, and Xa, with regard to their specific cofactors, tissue factor and factors VIIIa, Va, respectively. The methodological approach will consist of comparative active site mapping in the presence or absence of the cofactor. It is proposed to prepare series of peptide competitive inhibitors and substrates, in which the size and amino-acid composition will be systematically varied, and to measure their inhibition or kinetic constants in the presence or absence of the cofactor. The differential affinities thus determined should yield information on the sites where recognition is modified by the cofactor, i.e. the mechanism by which the cofactor may control the activity of the enzyme. Preliminary studies show that the effects of tissue factor on the interaction of factor VIIa with low molecular weight substrates and inhibitors could vary from minimal to considerable. The most significant effect in the presence of tissue factor was the enhancement of the reactivity of factor VIIa with transition-state analogues by 2-3 orders of magnitude. It is intended to learn whether this could be a common mode of action of coagulation factors on their enzymes, because, if correct, it can account in part for the dramatic contribution by the cofactors to the catalytic efficiency of the enzymes. A useful practical application of this work would be the design of potent and specific anti-thrombotic compounds.